PROJECT SUMMARY: Bacterial pneumonia kills more than 1 million newborns each year. Increased neonatal susceptibility to pneumonia is directly linked to immature infant lung mucosal defenses. Colonization by intestinal commensal bacteria, which begins immediately at birth, is hypothesized to be critical for postnatal development of neonate?s immune system, but the underlying mechanisms remain unclear. The premise of this proposal is that early life exposure to commensal bacteria promotes resistance to pneumonia in neonates by accelerating the immune cell development. Modern childbirth practices like increased use of antibiotics to treat preterm labor and cesarean deliveries alter the pattern of intestinal commensal colonization in the newborn and are associated with increased risk of pneumonia. Therefore, understanding this relationship has translational impact. This proposal is based on our recent publications, demonstrating that a rare population of sentinel immune cells- group 3 innate lymphoid cells (ILC3), are critical in defense against bacterial pneumonia in the newborn. A wave of ILC3 populates the murine and human lung in the postnatal period. ILC3 confer protection against bacterial pneumonia in newborn mice. Chemokine receptor, CCR4 was important for lung-specific trafficking of ILC3. This crosstalk was mediated by mucosal dendritic cells (DC), which capture the signals from intestinal commensal bacteria. Disruption of intestinal commensal bacteria with antibiotics abolished the expression of CCR4, interrupted the trafficking of ILC3 into the newborn?s lungs and rendered the antibiotic-treated neonatal mice susceptible to pneumonia in an interleukin (IL)-22 dependent fashion. These findings challenge the current paradigm that commensal bacteria-directed ILC3 development is locally restricted to the small intestine and support the hypothesis that postnatal colonization by intestinal commensal bacteria promotes resistance to pneumonia in neonates by accelerating the development of ILC3 in the newborn lung. The proposed experiments are designed to answer the following fundamental questions regarding the acquisition of pulmonary innate defenses in the newborn. 1) What is the migratory program of lung ILC3 in the newborns? 2) How do intestinal commensal bacteria instruct the migration of ILC3? 3) How do newly migrated ILC3 direct the neonatal pulmonary mucosal defenses against bacterial pneumonia? The proposed studies provide two conceptual advances regarding the development of pulmonary defense in the newborn. First, intestinal colonization by commensal bacteria is necessary for expansion of ILC3 in the newborn lung. Second, the newly expanded pool of ILC3 support lung epithelial stem cell proliferation and regulates pulmonary alveolar repair after pneumonia. These concepts could potentially suggest an alteration to the current practice of empiric broad-spectrum antibiotics in neonates. Our use of developmentally appropriate and clinically relevant murine model is complemented by a novel in vivo ILC3 expansion studies and alveolar organoids to probe ILC3-lung epithelial interactions. Finally, the proposed studies explore therapeutically relevant strategies, for example, commensal bacteria transfer to restore ILC3 development in antibiotic-treated newborns and use recombinant IL22 to promote alveolar repair. These studies will provide a framework to develop new therapeutic strategies to target ILC3 responses and promote lung mucosal defenses in newborns. !